This research evaluated serum amyloid A (SAA) concentration in synovial fluid of healthy horses as a potential marker for use in the diagnosis and monitoring of horses with septic arthritis. The first study evaluated the effect of arthroscopic lavage of healthy joints on concentrations of systemic and synovial SAA; as well as total protein concentration, nucleated cell count and percentage of neutrophils in synovial fluid. The second study, evaluated the effect of repeated through-and-through joint lavage on SAA in systemic blood and SAA, total protein, nucleated cell count and percentage of neutrophils in synovial fluid from healthy joints. In the first study, middle carpal joints of 6 horses were randomly assigned to one of the following treatments 1) arthrocentesis (controls) or 2) arthroscopic lavage. A washout period of 30 days was allowed in between treatments. Synovial fluid and blood samples were collected at 0, 24, 48, 72, 96 and 120 h. Measurements included SAA in blood and synovial fluid, and total protein, nucleated cell count and percentages of neutrophils in synovial fluid. In the second study, one tarsocrural joint was randomly assigned to receive repeated through-and-through joint lavage at 0, 48 and 96 h in 6 horses. Synovial fluid and blood samples were collected at 0, 24, 48, 72, 96 and 120 h. Measurements included SAA in blood and synovial fluid, and total protein, nucleated cell count and percentages of neutrophils in synovial fluid. For this study, synovial fluid samples collected at time 0 were considered as control values. After arthroscopic lavage and repeated through-and-through joint lavage, systemic and synovial SAA did not increase from baseline values (except for systemic SAA at 24h after arthroscopic lavage and in controls). Total protein values were significantly increased at all time points after arthroscopic and through-and-through joint lavages (except at 96h on both lavage procedures) but not in controls. With both lavage procedures, nucleated cell count significantly increased from baseline values at all time points (except at 96h after through-and-through joint lavage). Percentage of neutrophils was significantly increased after arthroscopic lavage at all time points and only at 24h in controls; however, the percentages of neutrophils were not significantly increased after repeated through-and-through joint lavage. Synovial SAA was not affected by arthroscopic or repeated through-and-through joint lavage; however, synovial total protein and nucleated cell counts were significantly increased. Synovial SAA may be a valuable inflammatory marker that is not affected by procedures as arthroscopic or repeated through-and-through joint lavage in horses. Further validation of synovial SAA as a marker for evaluating the progression of septic joints while treatment is installed is warranted.

A doença articular, especificamente a osteoartrite é uma das enfermidades mais prevalentes e mais debilitantes que acomete os cavalos, tendo um grande impacto econômico na indústria eqüina. Assim sendo, a investigação contínua e avanços na área terapêutica são de fundamental importância. A osteoartrite é uma doença degenerativa que pode ser deflagrada por uma série de fatores e onde, ultimamente, todos os tecidos articulares encontram-se comprometidos. Não obstante, é na degradação da matriz extracelular da cartilagem articular que ocorrem os eventos de maior expressão e repercussão. Na gênese da degradação da matriz extracelular encontra-se um desequilíbrio entre os processos anabólicos e catabólicos responsáveis pela homeostase normal da cartilagem articular e pela adaptação deste tecido às forças que sobre ele incidem. Estes processos são orquestrados por proteínas anabólicas, como, por exemplo o fator de crescimento tipo insulina 1 (IGF-l), e por citocinas inflamatórias que, de forma contrária, são responsáveis pela depleção de colágeno e de proteoglicanas da matriz, representando o grupo de proteínas catabólicas, cujo exemplo clássico é a interleucina-1. A interleucina-1 tem papel central nos processos fisiopatológicos da osteoartrite por desencadear vários eventos catabólicos nos sinoviócitos e condrócitos, incluindo a indução de gens de metaloproteinases e agrecanases e de outros mediadores inflamatórios como a cicloxigenase, a prostaglandina E2 e as espécies reativas do oxigênio. Seus efeitos biológicos se verificam após a ligação com dois tipos de receptores específicos e são modulados pela ocorrência natural de uma proteína antagonista destes receptores. O presente estudo procurou observar os efeitos antiinflamatórios desta proteína antagonista do receptor (IRAP) no líquido sinovial de equino através do emprego da técnica de citometria de fluxo. Nos ensaios observou-se que: 1-a adição de IRAP às células de líquido sinovial estimuladas in vitro por LPS e PMA reduziu a liberação de espécies reativas de oxigênio produzidas por elas; 2 - o plasma, quando utilizado como controle, exibiu efeitos semelhantes aos do IRAP sobre as células de líquido sinovial ativadas in vitro; 3- o efeito antiinflamatório deve-se mais à variação na intensidade de produção de espécies reativas do oxigênio do que à flutuação no percentil de células do líquido sinovial engajadas em sua geração, in vitro. Estes resultados suportam a aplicabilidade terapêutica do IRAP® pelo efeito antiinflamatório verificado sobre as células do liquido sinovial avaliadas por citometria de fluxo. Eles corroboram, ainda, para o uso da citometria de fluxo como instrumento eficaz na avaliação da produção de espécies reativas de oxigênio por células do líquido sinovial ativadas pelos estímulos de LPS e PMA, tanto quantitativamente como qualitativamente. / Joint disease in horses, specifically osteoarthritis, is one of the most prevalent and debilitating illnesses affecting equine industry and for this reason continued research and improvements in therapeutics are needed. Osteoarthritis is a degenerative disease that can be triggered by a number of factors and where ultimately all articular tissues are affected. The hallmark of osteoarthritis is the degeneration of the articular cartilage matrix, where the most relevant and expressive events take place. In the development of osteoarthritis there is disruption in extracellular matrix homeostasis with an overall balance toward cartilage metabolism. Homeostasis of the articular environment relies on balance between anabolic and catabolic events and results in ability of cartilage to respond to molecular or mechanical cues. This apparently antagonic processes are orquestrated by soluble protein mediators, for example the anabolic insulin-like growth factor-I (IGF-I), and, on the other side, by inflammatory cytokines, which in turn, are implicated in degradative processes of articular cartilage, characteristic of osteoarthritis. They deplete cartilage matrix from collagen and proteoglycans and the classical example of such a cytokine is interleukin-1. Interleukin-1 has a central role in the physiopathologic processes of osteoarthritis and has been implicated in the genesis of a number of catabolic events when acting on chondrocytes and synoviocytes. Examples are gene induction for metalloproteinases and agrecanases production, as well as production of other inflammatory mediators like ciclooxygenase, prostaglandin E2 and oxygen-derived reactive species. Its biological effects are observed after interaction with two different but specific types of receptors and are modulated by the occurrence of a natural antagonist, the interleukin-1 receptor antagonist protein (IRAP). In the present study the anti-inflammatory effects of this antagonist protein were evaluated in synovial fluid using cytometric flow techniques. It was observed that: 1- addition of IRAP to synovial fluid cells stimulated in vitro by LPS and PMA reduced the production of oxygen-derived reactive species; 2- plasma, used as a control, exhibited similar effects on activated synovial cells when compared to IRAP in vitro 3- the anti-inflammatory effect is due, in its majority, to the variation in intensity of oxygen-derived reactive species, more than on fluctuations on the percentage of synovial fluid cells actively engaged in its generation in vitro. These results support the therapeutic aplicability of IRAP® for its anti-inflammatory effect observed on synovial fluid cells evaluated with flow cytometric techniques. They also corroborate to the usefulness of cytometric flow techniques in equine synovial fluid cells; they are an invaluable tool to evaluate quantitative and qualitatively the production of oxygen-derived reactive species mediated by their activation with PMA and LPS.

Skeletal development begins when mesenchymal stem cells migrate, condensate and differentiate into chondrocytes. The chondrocytes differentiate in one of two ways. Either the cells form the cartilaginous template for endochondral ossification or they form the articular cartilage which express proteoglycan 4. The underlying mechanisms for articular cartilage formation are poorly understood. The purpose of this study was to assess the effect of different fractions of synoviocyte-conditioned medium on chondrocyte differentiation. We show evidence that Synovial-like fluid contains a protein which promotes chondrocytes to express proteoglycan 4, thus promoting articular cartilage formation. The synovial-like fluid was fractionized by size exclusion chromatography and reversed phase chromatography and thus, with that method, this manuscript lays the foundations for further research to identify the putative factor. Because of this study, we are now closer in identifying the proteins that promote articular cartilage formation.

Osteoarthritis (OA) causes debilitating pain for millions of people, yet OA is typically diagnosed late in the disease process after severe damage to the articular cartilage has occurred and few treatment options exist. Furthermore, destructive techniques are required to measure cartilage biochemical and mechanical properties for studying cartilage function and changes during OA. Hence, research and clinical needs exist for non-destructive measures of cartilage properties. Various arthroscopic (e.g., ultrasound probes) and imaging (e.g., MRI or CT) techniques are available for assessing cartilage less destructively. However, arthroscopic methods are limited by patient anesthesia/infection risks and cost, and MRI is hindered by high cost, long image acquisition times and low resolution. Contrast-enhanced CT (CECT) is a promising diagnostic tool for early-stage OA, yet most of its development work utilizes simplified and ideal cartilage models, and rarely intact, pre-clinical animal or human models. To advance CECT imaging for articular cartilage, this dissertation describes further development of a new cationic contrast agent (CA4+) for minimally-invasive assessment of cartilage biochemical and mechanical properties, including glycosaminoglycan content, compressive modulus, and coefficient of friction. Specifically, CA4+ enhanced CT is compared to these three cartilage properties initially using an ideal bovine osteochondral plug model, then the technique is expanded to examine human finger joints and both euthanized and live mouse knees. Furthermore, CECT attenuations with CA4+ map bovine meniscal GAG content and distribution, signifying CECT can evaluate multiple tissues involved in OA. CECT's sensitivity to critical cartilage and meniscal properties demonstrates its applicability as both a non-destructive research tool as well as a method for diagnosing and monitoring early-stage OA. Additionally, CECT enables evaluation of efficacy for a new biolubricant (2M TEG) for early-stage OA treatment. In particular, CECT can detect the reduced wear on cartilage surfaces for samples tested in 2M TEG compared to samples tested in saline (negative control). With its sensitivity to cartilage GAG content, surface roughness, and mechanical properties, CA4+ enhanced CT will serve as a valuable tool for subsequent in vivo animal and clinical use.

Diversos experimentos em humanos e equinos discutem a eficácia do uso intra-articular do ácido hialurônico (AH). Pouco se sabe a respeito de seu potencial pró ou anti-inflamatório, bem como sobre a eficácia das moléculas de AH de diferentes pesos moleculares. O objetivo deste estudo foi avaliar os efeitos anti-inflamatórios, anabólicos e antioxidantes da aplicação intra-articular de AH em sinovite aguda induzida por lipopolissacarídeo (LPS) em equinos. Foram utilizadas 24 articulações metacarpofalangeanas hígidas de 12 equinos jovens, avaliadas por exame físico, radiográfico e ultrassonográfico. As articulações foram divididas aleatoriamente em três grupos de oito, sendo o grupo controle tratado com triancinolona (TA) (10 mg), o grupo AH de baixo peso molecular (AH-BPM), e o grupo AH de alto peso molecular (AH-APM). Inicialmente, foram aplicados 0,25 ng de LPS por via intra-articular (momento 0). Após uma hora (momento 1), coletou-se líquido sinovial (LS) para contagem celular e imediatamente após realizado o tratamento com TA, AH-BPM ou AH-APM. Posteriormente, LS foi coletado nos momentos 8, 24 e 48 horas para contagem celular, avaliação do burst oxidativo, quantificação de prostaglandina E2 (PGE2), condroitim sulfato (CS), AH e mensuração do peso molecular do AH. Exame físico dos animais, incluindo mensuração da circunferência articular e avaliação de claudicação por meio do equipamento Lameness Locator® foi realizado em todos os momentos. As frequências cardíaca e respiratória dos animais aumentaram em todos os grupos às 8 horas (P<0,05), sendo menor no grupo TA. Em relação à circunferência articular o grupo AH-BPM apresentou maior aumento às 24 e 48 horas em relação aos outros grupos (p<0,05). O grau de claudicação do grupo AHAPM foi maior que o do grupo TA às 8 e 24 horas, mas similar ao grupo AH-BPM. O grupo TA apresentou maior quantidade de células nucleadas no LS do que o grupo AH-BPM as 24 e 28 horas e do que o grupo AH-APM às 48 horas. A concentração de PGE2 foi menor no grupo TA em relação ao grupo AH-APM às 8 horas, e a ambos os grupos que receberam AH às 48 horas (p<0,05). O grupo AH-APM mostrou maior concentração de AH às 8 horas do que os outros grupos, contudo às 48 horas o grupo TA mostrou maior concentração de AH do que os outros grupos (p<0,05). Também às 48 horas o grupo TA foi o que apresentou a menor porcentagem de AH de alto peso molecular (p<0,05). A maior concentração de CS no LS às 24 e 48 horas ocorreu no grupo TA, também observou-se o grupo TA foi o único em que a concentração de CS aumentou as 8 horas em relação aos valores basais. O grupo AH-APM foi o que apresentou menor concentração de CS às 8 horas (p<0,05). O grupo tratado com AHBPM apresentou maior intensidade de fluorescência nos momentos 24 e 48 horas (p<0,05). Apesar da triancinolona possuir efeito superior ao ácido hialurônico no controle da inflamação, ela possui efeito catabólico sobre a cartilagem articular, demonstrado pelo aumento da concentração de CS, além de quebra da molécula de AH no líquido sinovial. O AH-APM é mais indicado para terapia intra-articular em equinos uma vez que demonstrou menor efeito catabólico e oxidativo do que o AH-BPM / Several experiments have compared the efficacy of intra-articular usage of yaluronic acid (HA) within horses. Little is known about the effectiveness of different subtypes of HA in terms of its pro- and anti-inflammatory potential. The goal of this study was to investigate the anti-inflammatory, anabolic and antioxidant effects of intra-articular application of HA in acute synovitis induced by lipopolysaccharide (LPS) in horses. 24 metacarpophalangeal joints of 12 young horses without musculoskeletal alterations were subjected to lameness examination (Lameness Locator®), radiography and ultrasound. These joints were divided into three groups: a positive control group treated with triamcinolone acetonide (TA) (10mg) and 2 experimental groups treated with either hyaluronic acid of low molecular weight (HALMW) or hyaluronic acid of high molecular weight (HA- HWM). Initially, all joints were injected with 0.25ng of LPS. After an hour, synovial fluid (SF) was collected for cell counting. TA, HA- HWM or HA- LMW treatments were then applied to the joints. After treatment applications, SF was collected at 3 different time points (8, 24 and 48 hours) for cell counts, oxidative burst evaluation, prostaglandin E2 (PGE2) quantification, chondroitin sulfate (CS), HA and molecular weight measurements. Physical examinations, circumference measurements and lameness evaluations were carried out at each time point. The heart and respiratory rate of the animals increased in all groups at 8 hours (p <0.05), that increase was lower in the TA group. Regarding the circumference of the joint the HA-LMW group showed a greater increase at 24 and 48 hours compared to the other groups (p <0.05). The lameness of the HA-HMW group was higher than the TA group at 8 and 24 hours, but similar to the HALMW group. The TA group had a higher nucleated cells count in the SF compared to the HALMW group at 24 hours and 28 hours, and higher than the HA-HMW group at 48 hours. The PGE2 concentration was lower in the TA group in relation to the HA-HMW group at 8 hours, and lower than both groups that received HA at 48 hours (p <0.05). The HA-HMW group showed higher HA concentration at 8 hours than the other groups at 48 hours. However, the TA group showed higher HA concentration than the other groups (p <0.05). Also, at 48 hours the TA group was the one with the lowest percentage of high molecular weight HA (p <0.05). The highest concentration of CS in SF at 24 and 48 hours occurred in the TA group, and it was also observed in the TA group was the only one in which the concentration of CS increased at eight hours compared to baseline. The HA-HMW group showed the lowest concentration of CS at 8 hours (p <0.05). The group treated with HA-LMW showed higher fluorescence intensity at 24 and 48 hours (p <0.05). Although triamcinolone have an superior effect of hyaluronic acid in controlling inflammation, it has catabolic effects on cartilage, shown by increased concentration of CS, and breakage of the HA molecule in synovial fluid. The HA-HMW is recommended for intra-articular therapy in equine joints since it showed less oxidative and catabolic effects

Dentre as afecções que acometem as articulações dos equinos, a artrite séptica e a mais grave observada. A técnica da perfusão regional e um método comprovadamente eficiente para o tratamento de equinos acometidos por infecções sinoviais. O dimetilsulfóxido (DMSO) é um líquido orgânico, que possui a capacidade de penetrar, com extrema facilidade, em órgãos, tecidos e membranas celulares e intracelulares. Neste estudo, objetivou-se avaliar a concentração intra-articular da gentamicina administrada por perfusão regional intravenosa (PRI), associada ou não ao DMSO, bem como avaliar a influência do volume total perfundido o período de tempo onde a concentração inibitória mínima (CIM) foi mais eficiente e se a associação com o DMSO aumentou a CIM no líquido sinovial. Os animais foram distribuídos em quatro grupos experimentais, cada grupo recebeu gentamicina 6,6 mg/kg por PRI, o volume a ser administrado, após o cálculo da quantidade de gentamicina acrescida ou não de DMSO, era completado com solução de ringer com lactato estéril até o volume de 60mL nos grupos G60 e GD60, e até 250mL para os grupos G250 e GD250. As colheitas de líquido sinovial foram realizadas antes do início do experimento (T0), imediatamente depois da retirada do torniquete (T1) e após 4 (T2), 8 (T3), 12 (T4), 16 (T5) e 24 (T6) horas. O método para doseamento das concentrações de gentamicina empregado foi o de difusão em ágar. Destacamos que as concentrações de gentamicina no líquido sinovial na dose de 6.6.mg/Kg podem ser consideradas como adequadas, num período de até 24 horas após a administração. Nossos resultados apontam que o volume de 60 mL, pode ser considerado como o volume ideal de perfusão, bem como a associação do DMSO aumentou as concentrações de gentamicina (µg/mL) na articulação dos equinos e possivelmente reduziu a formação de edemas e aumentos de volume locais. / Among the diseases that affect the joints of horses, septic arthritis is the most serious observed. The regional perfusion technique is a well proven method for the treatment of horses affected by synovial infections. Dimethylsulfoxide (DMSO) is an organic liquid that has the ability to penetrate, with extreme ease on organs, tissues and cellular and intracellular membranes. This study aimed to evaluate the intra-articular concentration of gentamicin administered by intravenous regional perfusion (PRI), associated or not with DMSO, and assess the influence of the total volume infused the time period in which the minimum inhibitory concentration (MIC) It was more efficient and the association with DMSO increased the CIM in the synovial fluid. The animals were divided into four groups, each group received 6.6 mg gentamicin / kg per PRI, the volume to be administered, after calculating the amount of gentamicin plus or absence of DMSO was completed with Ringer\'s lactate solution with sterile until the volume in 60mL groups G60 and GD60, and up to 250 mL and G250 GD250 groups. The synovial fluid samples were collected before the start of the experiment (T0), immediately after removal of the tourniquet (T1) and after 4 (T2), 8 (T3), 12 (T4), 16 (T5) and 24 (T6) hours. The method for determination of gentamicin concentrations was employed the agar diffusion. We emphasize that the gentamicin concentrations in synovial fluid in 6.6.mg/Kg dose may be considered suitable, a period of up to 24 hours after administration. Our results indicate that the volume of 60 ml, can be considered as the ideal volume of the infusion, as well as the association of DMSO increased the gentamicin concentrations (µg / ml) in the joint of horses and possibly reduced edema formation and increases local volume.

A doença articular, especificamente a osteoartrite é uma das enfermidades mais prevalentes e mais debilitantes que acomete os cavalos, tendo um grande impacto econômico na indústria eqüina. Assim sendo, a investigação contínua e avanços na área terapêutica são de fundamental importância. A osteoartrite é uma doença degenerativa que pode ser deflagrada por uma série de fatores e onde, ultimamente, todos os tecidos articulares encontram-se comprometidos. Não obstante, é na degradação da matriz extracelular da cartilagem articular que ocorrem os eventos de maior expressão e repercussão. Na gênese da degradação da matriz extracelular encontra-se um desequilíbrio entre os processos anabólicos e catabólicos responsáveis pela homeostase normal da cartilagem articular e pela adaptação deste tecido às forças que sobre ele incidem. Estes processos são orquestrados por proteínas anabólicas, como, por exemplo o fator de crescimento tipo insulina 1 (IGF-l), e por citocinas inflamatórias que, de forma contrária, são responsáveis pela depleção de colágeno e de proteoglicanas da matriz, representando o grupo de proteínas catabólicas, cujo exemplo clássico é a interleucina-1. A interleucina-1 tem papel central nos processos fisiopatológicos da osteoartrite por desencadear vários eventos catabólicos nos sinoviócitos e condrócitos, incluindo a indução de gens de metaloproteinases e agrecanases e de outros mediadores inflamatórios como a cicloxigenase, a prostaglandina E2 e as espécies reativas do oxigênio. Seus efeitos biológicos se verificam após a ligação com dois tipos de receptores específicos e são modulados pela ocorrência natural de uma proteína antagonista destes receptores. O presente estudo procurou observar os efeitos antiinflamatórios desta proteína antagonista do receptor (IRAP) no líquido sinovial de equino através do emprego da técnica de citometria de fluxo. Nos ensaios observou-se que: 1-a adição de IRAP às células de líquido sinovial estimuladas in vitro por LPS e PMA reduziu a liberação de espécies reativas de oxigênio produzidas por elas; 2 - o plasma, quando utilizado como controle, exibiu efeitos semelhantes aos do IRAP sobre as células de líquido sinovial ativadas in vitro; 3- o efeito antiinflamatório deve-se mais à variação na intensidade de produção de espécies reativas do oxigênio do que à flutuação no percentil de células do líquido sinovial engajadas em sua geração, in vitro. Estes resultados suportam a aplicabilidade terapêutica do IRAP® pelo efeito antiinflamatório verificado sobre as células do liquido sinovial avaliadas por citometria de fluxo. Eles corroboram, ainda, para o uso da citometria de fluxo como instrumento eficaz na avaliação da produção de espécies reativas de oxigênio por células do líquido sinovial ativadas pelos estímulos de LPS e PMA, tanto quantitativamente como qualitativamente. / Joint disease in horses, specifically osteoarthritis, is one of the most prevalent and debilitating illnesses affecting equine industry and for this reason continued research and improvements in therapeutics are needed. Osteoarthritis is a degenerative disease that can be triggered by a number of factors and where ultimately all articular tissues are affected. The hallmark of osteoarthritis is the degeneration of the articular cartilage matrix, where the most relevant and expressive events take place. In the development of osteoarthritis there is disruption in extracellular matrix homeostasis with an overall balance toward cartilage metabolism. Homeostasis of the articular environment relies on balance between anabolic and catabolic events and results in ability of cartilage to respond to molecular or mechanical cues. This apparently antagonic processes are orquestrated by soluble protein mediators, for example the anabolic insulin-like growth factor-I (IGF-I), and, on the other side, by inflammatory cytokines, which in turn, are implicated in degradative processes of articular cartilage, characteristic of osteoarthritis. They deplete cartilage matrix from collagen and proteoglycans and the classical example of such a cytokine is interleukin-1. Interleukin-1 has a central role in the physiopathologic processes of osteoarthritis and has been implicated in the genesis of a number of catabolic events when acting on chondrocytes and synoviocytes. Examples are gene induction for metalloproteinases and agrecanases production, as well as production of other inflammatory mediators like ciclooxygenase, prostaglandin E2 and oxygen-derived reactive species. Its biological effects are observed after interaction with two different but specific types of receptors and are modulated by the occurrence of a natural antagonist, the interleukin-1 receptor antagonist protein (IRAP). In the present study the anti-inflammatory effects of this antagonist protein were evaluated in synovial fluid using cytometric flow techniques. It was observed that: 1- addition of IRAP to synovial fluid cells stimulated in vitro by LPS and PMA reduced the production of oxygen-derived reactive species; 2- plasma, used as a control, exhibited similar effects on activated synovial cells when compared to IRAP in vitro 3- the anti-inflammatory effect is due, in its majority, to the variation in intensity of oxygen-derived reactive species, more than on fluctuations on the percentage of synovial fluid cells actively engaged in its generation in vitro. These results support the therapeutic aplicability of IRAP® for its anti-inflammatory effect observed on synovial fluid cells evaluated with flow cytometric techniques. They also corroborate to the usefulness of cytometric flow techniques in equine synovial fluid cells; they are an invaluable tool to evaluate quantitative and qualitatively the production of oxygen-derived reactive species mediated by their activation with PMA and LPS.

A cartilagem articular é um tecido avascular, com baixa celularidade, composta principalmente de colágeno extracelular e proteoglicanos, com uma capacidade limitada de regeneração. Nos últimos anos, diversas abordagens clínicas e de pesquisa têm sido adotadas para reparar danos na cartilagem articular, como transplante de condrócitos, enxerto de periósteo, células-tronco mesenquimais e tecidos derivados dessas células. O isolamento de células-tronco mesenquimais foi relatado a partir de diferentes tecidos, incluindo medula óssea, tecido adiposo, sangue do cordão umbilical, sangue periférico e saco vitelino em equinos. As células-tronco mesenquimais derivadas de líquido e membrana sinoviais foram obtidas em humanos, cães, suínos e caprinos e são fontes promissoras para regeneração articular, já que são tecido-específicas e de fácil obtenção e cultivo. O objetivo deste trabalho foi estabelecer e caracterizar a linhagens de células obtidas de membrana e líquido sinoviais equinos. Os fragmentos foram obtidos por meio de artroscopias e cultivados em meios de cultura DMEM-H e MEM para obtenção das linhagens. Para análise da morfologia celular foi realizada a fotodocumentação das garrafas em microscopia invertida. A expressão de marcadores de células-tronco (CD45RO, OCT3/4, NANOG, CD105, CD90, CD34, CD117, CD133, TRA-1-81, VEGF-R1 e Ly6a), marcadores inflamatórios (COX-2, TNF-R1-r, CD11, CD1a e MCP-1) e marcadores envolvidos na checagem e progressão do ciclo celular (Caspase-3 fosforilada, HSP-47, P21, Ki67, Ciclina D1 e P53) mostraram diferencialmente expressos, sugerindo que podemos considerá-las uma possível fonte de células-tronco mesenquimais. Devido ao grande impacto que patologias na articulação têm sobre o desempenho atlético em cavalos, os resultados demonstrados neste trabalho será uma base para conduzir outros experimentos na avaliação das aplicações terapêuticas dessas células. / The articular cartilage is an avascular tissue with low cellularity composed of extracellular collagen and proteoglicans. It has a limited capacity of regeneration. Condrocyte transplantation, mesenchymal stem cells and tissues derived from these cells has been used by several researches to repair damage to the articular cartilage. The isolation of mesenchymal stem cells has been reported from different tissues such as bone marrow, adipose tissue, blood, umbilical cord, and yolk sac. The mesenchymal stem cells from synovial fluid and synovial membrane were obtained from humans, dogs, pigs and goats. These cells are tissue specific and easy to obtain and cultivate. The objective of this research is to obtain and characterize cells from equine synovial fluid and synovial membrane. The samples were obtained by arthroscopy and cultivated in the DMEM-H and MEM media. Cell morphology analyses were made by photodocumentation in inverted microscopy. The expression of stem cell markers (CD45RO, OCT3/4, NANOG, CD105, CD90, CD34, CD117, CD133, TRA-1-81, VEGF-R1 and Ly6a), inflammation markers (COX-2, TNF-R1-r, CD11, CD1a and MCP-1) and markers involved in checking and cell cycle progression (Caspase-3, HSP-47, P21, Ki67, Ciclina D1 and P53) showed differentially expressed. Mesenchymal stem cells from synovial membrane and synovial fluid provide promise for cell-based therapies for articular cartilage repair. These results may lead other experiments to use these cells to new therapeutic applications.

Articular cartilage is an avascular, aneural, and alymphatic tissue with a structure consisting of a superficial, a middle and a deep zone, overlie a calcified zone at the cartilage border between. Each zone has biological and mechanical properties. Self-repair of damaged cartilage seldom if ever occurs, and joint injuries that harm cartilage surfaces often result in osteoarthritis. This has prompted researchers to explore diverse approaches to cartilage regeneration. The superficial zone shows the highest cellularity and the lowest matrix density. Cartilage cells (chondrocytes) residing in the superficial zone had been thought to be a subpopulation of chondrocytes. However, our laboratory identified a second population of cells that were distinguishable from chondrocytes based on their clonogenicity, multipotency, migratory activity, higher proliferate rate and substantial morphological differences. These cells later proved to be chondrogenic progenitor cells (CPCs). Our continuing studies have shown that CPCs are less chondrogenic than normal chondrocytes and their function is to protect the cartilage surface rather than to regenerate cartilage matrix as previously supposed. In addition, we found evidence to suggest that CPCs act as pro-inflammatory cells in the context of cartilage injury. For these reasons, we undertook a more comprehensive comparison of the phenotypic differences between CPCs and normal chondrocytes and between CPCs and joint cells (tissue synoviocytes from the joint capsule and cells present in synovial fluid) which have been shown to be play roles in joint inflammation. Gene expression microarray analysis of >25,000 genes revealed that the overall pattern of gene expression in CPCs was distinct from normal chondrocytes, but closely related to synoviocytes and synovial fluid cells. Analysis of specific genes by quantitative PCR (qPCR) showed profound differences between CPCs and normal chondrocytes in terms of cartilage matrix gene expression (Collagen Type ІІ, Aggrecan, Link Protein and COMP) and pro-inflammatory gene expression (IL6, IL8, CCL2 and CXCL12). In contrast, the pattern of CPC gene expression closely resembled. Sulfated glycosaminoglycan assays revealed that cartilage matrix deposition by CPCs, as well as synoviocytes and synovial fluid cells, was significantly inferior to normal chondrocytes. However, chondrogenic and osteogenic differentiation assays, showed no significant differences among the four cell types. In addition to establishing that CPCs are distinct from chondrocytes, this work suggests significant revisions to our understanding of CPC function in cartilage. The weak chondrogenic ability and higher expression of inflammatory cytokines, suggests these cells don't play a regenerative role as previously thought. On the other, we found evidence that CPCs may form a protective layer on the top of the injured cartilage surfaces, preventing further cartilage injury. In vivo studies are needed to fully elucidate the significance of these roles in cartilage health and disease.

The composition of orthopaedic wear testing lubricants used to mimic synovial fluid (SF) is known to significantly affect in vitro polyethylene (PE) wear; however, some wear testing standards may be promoting the use of lubricants that are not clinically relevant. The present thesis evaluated the biochemical composition of human osteoarthritic and periprosthetic SF in order to propose changes to lubricant specifications in current wear testing standards. Using this data, pin-on-disc wear tests were conducted to explore the effects of more clinically relevant lubricants on PE wear. Results showed that wear decreased using a more clinically relevant lubricant. Samples of these lubricants were biochemically evaluated and compared to the SF results previously obtained, which showed that current standards for wear testing lubricants are biochemically different from SF. The findings from the present thesis encourage the modification of standardized lubricant specifications to improve wear testing protocols and guarantee clinically relevant wear testing.